Discovery of CLC transport proteins: cloning, structure, function and pathophysiology

Abstract

After providing a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl(-) channel ClC-0 and the discovery of the CLC gene family, I succinctly describe the general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl(-) channels and vesicular Cl(-) /H(+) -exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel.

Figure 1

Cloning of the Torpedo channel ClC-0 by hybrid depletion Total electric organ RNA, which was hybrid-depleted with single-stranded DNA derived from pools of 12 clones from a highly size-selected cDNA library, was expressed in Xenopus oocytes. Current ‘fingerprints’ were obtained using a symmetrical voltage clamp-protocol (A, inset) and recorded by a chart recorder. After the current response had increased to steady-state magnitudes (as a result of opening of the slow gate), the response to low chloride was recorded at depolarizing potentials. Subsequent superfusion with acetylcholine (ACh) probed for the expression of the Torpedo AChR that was used as internal reference to avoid false positives as a result of RNA degradation. A, background currents in non-injected oocytes; no response to ACh. B, negative pool of clones that shows normal Cl⁻ channel and AChR expression. C, positive pool containing a partial ClC-0 cDNA; reduction of Cl⁻ current with normal response to ACh. D, expression of full-length ClC-0 cRNA; large Cl⁻ currents and no response to ACh. Oocytes were measured in ND96 (in mm: 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂), except for the low chloride pulse (7 mm Cl⁻). AChR currents were elicited by 1 mm acetylcholine in the presence of 10 μm atropine to block muscarinic receptors. Modified from Jentsch et al. (1990).

Figure 2

Modelling vesicular acidification with Cl⁻ channels and Cl⁻/H⁺-exchangers Reductionist model calculations revealing differential effects of Cl⁻ channels or 2 Cl⁻/H⁺-exchangers on the acidification of vesicles (modified from Weinert et al. (2010)). ATP is virtually added at t = 0. (−) model vesicles containing only a proton pump and a proton leak nearly instantaneously reach a high luminal potential (B) that is given by the energy supplied by ATP hydrolysis. Virtually no acidification occurs (A). (unc) model vesicles containing additionally a Cl⁻ channel, as in the classical model of vesicular acidification and as realized in Clcn5^unc/y and Clcn7^unc/unc mice (Novarino et al. ; Weinert et al. 2010), acidify their lumen (A) and accumulate Cl⁻ (C). They reach a more moderate inside-positive potential (B). (WT) model vesicles containing instead of a Cl⁻ channel a 2 Cl⁻/H⁺-exchanger (CLC antiport) rather unexpectedly reach a more acidic steady-state pH than those containing a Cl⁻ channel (A). This is related to the fact that they reach a more negative luminal potential (B). They also accumulate more Cl⁻ (C), as expected from H⁺-diven uptake of Cl⁻. For equations and parameter used, see Weinert et al. (2010).

Figure 3

Role of ClC-K/barttin channels in transepithelial transport Schematic diagram of NaCl reabsorption in the TAL of Henle’s loop (A) and of K⁺ secretion by marginal cells in the stria vascularis of the inner ear (B) (Taken from Estévez et al. (2001)).